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Synthetic human cell fate regulation by protein-driven RNA switches.

Saito H, Fujita Y, Kashida S, Hayashi K, Inoue T - Nat Commun (2011)

Bottom Line: Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways.A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression.The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Gene Biodynamics, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. [2] International Cooperative Research Project, Japan Science and Technology Agency, 5 Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan. [3] The Hakubi Center, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT
Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

No MeSH data available.


Protein-driven ON system.(a) Schematic illustration of the ON/OFF states. (b) Sh-GFP targeting the EGFP gene. The sequence in red is complementary to EGFP mRNA (1). Sh-N was designed not to knockdown any gene and encodes a stop codon (in yellow) in every frame (2). We designed Kt-Sh-GFP by incorporating the Kt motif into the loop region of the Sh-GFP (3). dKt-Sh-GFP is defective for the Kt motif in the loop region (4). (c) Determination of the binding affinity of Kt-Sh-GFP (40 nM) and L7Ae (0–640 nM) using the gel shift assay. Kt-Sh-GFP and L7Ae were mixed in transfection buffer, Opti-MEMI (Invitrogen), and separated in the gel shift assay. (d) The specific binding of Kt and L7Ae repressed Dicer cleavage activity for Kt-Sh-GFP. In the presence of L7Ae, Kt-Sh-GFP and L7Ae formed an RNP complex and prevented Dicer cleavage, yet the mutant dKt-Sh-GFP was still cleaved. (e) The relative concentration of EGFP mRNA. (f) The intensity of EGFP fluorescence in HeLa cells stably expressing EGFP (HeLa-GFP). These indicated that L7Ae-specific binding to Kt-Sh-GFP repressed the knock down function of Kt-Sh-GFP and, as a result, inhibited EGFP mRNA degradation and reactivated EGFP expression in HeLa-GFP cells. For e and f, the error bar indicates the standard deviation of three independent samples. Sh-GFP and Sh-N were used for positive and negative controls of EGFP knockdown, respectively.
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f3: Protein-driven ON system.(a) Schematic illustration of the ON/OFF states. (b) Sh-GFP targeting the EGFP gene. The sequence in red is complementary to EGFP mRNA (1). Sh-N was designed not to knockdown any gene and encodes a stop codon (in yellow) in every frame (2). We designed Kt-Sh-GFP by incorporating the Kt motif into the loop region of the Sh-GFP (3). dKt-Sh-GFP is defective for the Kt motif in the loop region (4). (c) Determination of the binding affinity of Kt-Sh-GFP (40 nM) and L7Ae (0–640 nM) using the gel shift assay. Kt-Sh-GFP and L7Ae were mixed in transfection buffer, Opti-MEMI (Invitrogen), and separated in the gel shift assay. (d) The specific binding of Kt and L7Ae repressed Dicer cleavage activity for Kt-Sh-GFP. In the presence of L7Ae, Kt-Sh-GFP and L7Ae formed an RNP complex and prevented Dicer cleavage, yet the mutant dKt-Sh-GFP was still cleaved. (e) The relative concentration of EGFP mRNA. (f) The intensity of EGFP fluorescence in HeLa cells stably expressing EGFP (HeLa-GFP). These indicated that L7Ae-specific binding to Kt-Sh-GFP repressed the knock down function of Kt-Sh-GFP and, as a result, inhibited EGFP mRNA degradation and reactivated EGFP expression in HeLa-GFP cells. For e and f, the error bar indicates the standard deviation of three independent samples. Sh-GFP and Sh-N were used for positive and negative controls of EGFP knockdown, respectively.

Mentions: Like the OFF system, the protein-triggered translational activation system (ON system) should work as a 'protein–protein information converter' that transmits information carried by an input protein (for example, L7Ae) to the desired output protein (for example, Bcl-xL). Such converters are useful for bypassing intrinsic cell signalling pathways or constructing synthetic signalling pathways (Fig. 1)1920. To construct the ON system in human cells, we designed and constructed a synthetic shRNA293031 containing an siRNA and the Kt motif in its stem and loop regions, respectively (Figs 1 and 3a; Supplementary Fig. S6). We will refer to this construct as Kt-shRNA. In this system, the Kt-shRNA is designed to result in specific degradation of a target mRNA by RNA interference (RNAi). Conversely, cellularly expressed L7Ae binding to Kt-shRNA is expected to interfere with Dicer-dependent cleavage of the shRNA, resulting in L7Ae-controlled protection of the target mRNA (Fig. 3a).


Synthetic human cell fate regulation by protein-driven RNA switches.

Saito H, Fujita Y, Kashida S, Hayashi K, Inoue T - Nat Commun (2011)

Protein-driven ON system.(a) Schematic illustration of the ON/OFF states. (b) Sh-GFP targeting the EGFP gene. The sequence in red is complementary to EGFP mRNA (1). Sh-N was designed not to knockdown any gene and encodes a stop codon (in yellow) in every frame (2). We designed Kt-Sh-GFP by incorporating the Kt motif into the loop region of the Sh-GFP (3). dKt-Sh-GFP is defective for the Kt motif in the loop region (4). (c) Determination of the binding affinity of Kt-Sh-GFP (40 nM) and L7Ae (0–640 nM) using the gel shift assay. Kt-Sh-GFP and L7Ae were mixed in transfection buffer, Opti-MEMI (Invitrogen), and separated in the gel shift assay. (d) The specific binding of Kt and L7Ae repressed Dicer cleavage activity for Kt-Sh-GFP. In the presence of L7Ae, Kt-Sh-GFP and L7Ae formed an RNP complex and prevented Dicer cleavage, yet the mutant dKt-Sh-GFP was still cleaved. (e) The relative concentration of EGFP mRNA. (f) The intensity of EGFP fluorescence in HeLa cells stably expressing EGFP (HeLa-GFP). These indicated that L7Ae-specific binding to Kt-Sh-GFP repressed the knock down function of Kt-Sh-GFP and, as a result, inhibited EGFP mRNA degradation and reactivated EGFP expression in HeLa-GFP cells. For e and f, the error bar indicates the standard deviation of three independent samples. Sh-GFP and Sh-N were used for positive and negative controls of EGFP knockdown, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105309&req=5

f3: Protein-driven ON system.(a) Schematic illustration of the ON/OFF states. (b) Sh-GFP targeting the EGFP gene. The sequence in red is complementary to EGFP mRNA (1). Sh-N was designed not to knockdown any gene and encodes a stop codon (in yellow) in every frame (2). We designed Kt-Sh-GFP by incorporating the Kt motif into the loop region of the Sh-GFP (3). dKt-Sh-GFP is defective for the Kt motif in the loop region (4). (c) Determination of the binding affinity of Kt-Sh-GFP (40 nM) and L7Ae (0–640 nM) using the gel shift assay. Kt-Sh-GFP and L7Ae were mixed in transfection buffer, Opti-MEMI (Invitrogen), and separated in the gel shift assay. (d) The specific binding of Kt and L7Ae repressed Dicer cleavage activity for Kt-Sh-GFP. In the presence of L7Ae, Kt-Sh-GFP and L7Ae formed an RNP complex and prevented Dicer cleavage, yet the mutant dKt-Sh-GFP was still cleaved. (e) The relative concentration of EGFP mRNA. (f) The intensity of EGFP fluorescence in HeLa cells stably expressing EGFP (HeLa-GFP). These indicated that L7Ae-specific binding to Kt-Sh-GFP repressed the knock down function of Kt-Sh-GFP and, as a result, inhibited EGFP mRNA degradation and reactivated EGFP expression in HeLa-GFP cells. For e and f, the error bar indicates the standard deviation of three independent samples. Sh-GFP and Sh-N were used for positive and negative controls of EGFP knockdown, respectively.
Mentions: Like the OFF system, the protein-triggered translational activation system (ON system) should work as a 'protein–protein information converter' that transmits information carried by an input protein (for example, L7Ae) to the desired output protein (for example, Bcl-xL). Such converters are useful for bypassing intrinsic cell signalling pathways or constructing synthetic signalling pathways (Fig. 1)1920. To construct the ON system in human cells, we designed and constructed a synthetic shRNA293031 containing an siRNA and the Kt motif in its stem and loop regions, respectively (Figs 1 and 3a; Supplementary Fig. S6). We will refer to this construct as Kt-shRNA. In this system, the Kt-shRNA is designed to result in specific degradation of a target mRNA by RNA interference (RNAi). Conversely, cellularly expressed L7Ae binding to Kt-shRNA is expected to interfere with Dicer-dependent cleavage of the shRNA, resulting in L7Ae-controlled protection of the target mRNA (Fig. 3a).

Bottom Line: Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways.A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression.The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Gene Biodynamics, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. [2] International Cooperative Research Project, Japan Science and Technology Agency, 5 Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan. [3] The Hakubi Center, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT
Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

No MeSH data available.